PMC

CTCF sites A and C of Tsix are differentially methylated in gametes. (A) MSRA at sites A and C using conventional PCR. (B) Representative MSRA of at site A using quantitative real-time PCR analysis. Each curve represents the amplification of a single sample. Horizontal orange lines, fluorescence threshold. HgaI-digested (red arrows) and undigested (black arrows) samples were run in triplicates. (C) Bisulfite analysis of site A. Filled circle, methylated; open circle, unmethylated. n, number of strands exhibiting each methylation pattern.

Mutant analyses suggest that DNA methylation works genetically upstream to Tsix and Xite. (A) The Tsix^ΔCpG and Xite^ΔL positions are shown. Proposed DMDs are indicated as filled black rectangles. (B) Effects of Xite^ΔL on CTCF sites A and C methylation at Tsix. MSRA analysis using real-time PCR of wild-type and mutant female cells lines at sites A and C. (C) Effects of Tsix^ΔCpG on Xite DMDs. MSRA analysis using real-time PCR of wild-type and mutant cell lines at the Xite DMDs. The deduced methylation status is shown. Female mutants are heterozygous. Open circles, unmethylated; filled circles, methylated.

Differential methylation at the Xite locus correlates with events of XCI. (A) Map of Xite. Methylation-sensitive restriction sites are indicated above the axis; amplicons are indicated below the axis. Black rectangles, novel DMDs. (B) MSRA using real-time PCR at Xite. The deduced methylation status is shown; open circle, unmethylated; filled circles, methylated; half-filled circles, partial methylation. ES cells are day 0 (d0) cultures, EB cells are from days 7 to 12. ND, no data. (C) Bisulfite analysis at DHS6. CpG residues examined are numbered across; the enzyme recognition sites are noted. Filled circle, methylated; open circle, unmethylated; n, number of strands exhibiting each methylation pattern. (D) Allele-specific MSRA of ES and EB cells at DHS6. Enzyme indicates samples that were digested (+) with either HpaII, AciI, or HgaI or undigested (−) before PCR amplification with primers NG-1 and NG-2 and then probing with end-labeled primer KHp388. (E) Allele-specific MSRA of morula and female TS cells at DHS6 as described in panel D. For the TS cells, the maternal allele is of 129 origin, the paternal allele of castaneus origin. For morulae, the maternal allele is of castaneus origin, and the paternal allele is of 129 origin.

Southern blot analysis of the Tsix region reveals potential DMDs. (A) Map of the Tsix and Xite regions. Oval region, CTCF motifs. DHS, DNase I-hypersensitive sites. Inset: H, HpaII; A, AciI. (B) Genomic Southern blot analysis of the 5′ end of Tsix using the combination of enzymes indicated. Probe, 1.1-kb AgeI-HincII fragment at the 5′ end of Tsix; Sp, sperm; M, male liver; F, female liver; filled circles, methylated bands; open circles, unmethylated bands. (C) Genomic Southern analysis of undifferentiated (day 0) ES cells and differentiated (day 12) EB cells using the 1.1-kb AgeI-HincII probe. Filled circles, methylated bands; open circles, unmethylated bands. Asterisks, doublet lower bands associated with the female ES and EB DNAs resulted from DXpas34 repeat length polymorphisms between the 129-derived and Mus castaneus-derived Xs in 16.7 ES cells. (D) Genomic Southern analysis of DXPas34 using the same probe. ES cells are female. The restriction sites (HpaII and AciI) are those indicated in the panel A inset. Asterisks indicate sites in ES cells that have become hypermethylated and have shifted upward and lost intensity or disappeared in EB cells.

Developmentally regulated methylation profiles at CTCF sites A and C. (A) Bisulfite analysis at site A. The methylation state of the single CpG is shown. (B) Bisulfite analysis at site C. CpG residues examined are numbered across; the AciI recognition site is noted. Filled circle, methylated; open circle, unmethylated; n, number of strands exhibiting each methylation pattern. (C) MSRA using real-time PCR of various cell lines and tissues. Filled circle, methylated; half-filled circle, partially methylated; open circle, unmethylated (see the text). ES cells are undifferentiated day 0 (d0) cultures; EB cells are differentiated cultures at days 7 to 12, a time when a vast majority of cells have undergone XCI (). (D) Representative real-time PCR runs of the MSRA performed in triplicates. Black arrows, undigested samples; red arrows, digested samples. (E) Allele-specific MSRA at site C in ES and EB cells. Samples were digested (+) or not digested (−) with AciI before PCR amplification with primers CC3-1B and CC3-1C and probing with end-labeled primer, KHp4.